Project Summary/Abstract It has been well established that periodontitis is exacerbated by metabolic syndrome (MetS). To understand the underlying mechanisms, we found that MetS-associated palmitic acid (PA) and periodontitis- related lipopolysaccharide (LPS) synergistically upregulate inflammatory cytokines in macrophages via acid sphingomyelinase-dependent ceramide generation (ASMase-CER pathway). Based on these findings, we are testing our hypothesis that ASMase is a potential target for MetS-exacerbated periodontitis. Currently, we are investigating how ASMase-CER pathway is involved in the osteoclast formation and fibroblast apoptosis. We are also investigating how ASMase-CER pathway mediates PA-amplified TLR4 signaling in macrophages. Thus, we have focused on characterizing the role of macrophages and fibroblasts in MetS-related periodontitis. However, recent studies have highlighted that gingival epithelial cells play key roles in the development of periodontitis. The gingival epithelium forms a unique structure that provides the first line of defense against pathogens. However, oral pathogens such as P. gingivalis can impact on the protective functions of the gingival epithelium. Furthermore, the gingival epithelium also expresses proinflammatory cytokines, which contribute to periodontitis. Therefore, to better understand the mechanisms by which MetS exacerbates periodontitis, it is essential to investigate the role of the gingival epithelium in MetS-exacerbated periodontitis. In this study, we proposed two specific aims: 1. To determine the role of the gingival epithelium in MetS-exacerbated periodontal inflammation in animal models and the potential involvement of ASMase-CER pathway in the MetS-periodontitis interaction. We hypothesized that MetS specifically augments P. gingivalis- modulated inflammatory response in the gingival epithelium via ASMase-CER pathway, which contributes to the exacerbated periodontitis. 2. To determine the effect of the crosstalk between P. gingivalis and saturated fatty acid (SFA) on the inflammatory response and the epithelial mesenchymal transition-associated molecular events in gingival epithelial cells in the context of ASMase-CER pathway. We hypothesized that MetS-related SFA promotes P. gingivalis-modulated inflammatory response of gingival epithelial cells and P. gingivalis- inhibited expression of epithelial integrity and function-related proteins via ASMase-CER pathway. In this research project, the PI will collaborate with Dr. Yilmaz, a clinician scientist and a microbiologist specialized in P. gingivalis and the P. gingivalis-epithelium interaction. In the collaboration, Dr. Yilmaz and her lab will provide expertise in the pathobiology of gingival epithelial cells and the applications of the animal model with P. gingivalis inoculation as well as cell and molecular biology techniques. We believe that this newly established collaboration between the PI and Dr. Yilmaz will yield novel insights into the pathogenesis of MetS- exacerbated periodontitis, which will significantly enhance the approved aims of the parent grant.